1. Field of the Invention
This invention relates to the attachment of detectable labels to molecules containing free amino groups (e.g., proteins, nucleic acids and amino sugars), the molecules having specific binding sites thereby providing a method of directing the label to the moiety specifically bound by the labeled molecule.
2. Review or Related Art
N-hydroxysulfosuccinimide (NHS) esters provide one of the most common activation chemistries for creating reactive acylating agents. Homobifunctional NHS esters were first introduced as reactive cross-linking agents in the early 1970""s and are widely available commercially. [Bragg, P., and Hou, C. (1975), xe2x80x9cSubunit composition, function and spatial arrangement on the Ca2+ and Mg2+ activated adenosine triphosphatases of Escherichia coli and Salmonella typhimurium,xe2x80x9d Eur. J Biochem., 106, 495-503, Lomant, A. and Fairbanks, G. (1976)J. Mol. Biol.,104, 243-261.] NHS esters are routinely used to conjugate proteins to each other via heterobifunctional cross-linking agents (e.g. see all of Pierce Chemicals xe2x80x9cDouble Agentsxe2x80x9d that have an NHS side; Muramoa K, Kamiya H (1988) xe2x80x9cPreparation and characterization of a cleavable photoactiviable heterobifunctional fluoresent reagent for proteins.xe2x80x9d Agric. Biol Chem. 52, 547-554) or to dyes (such as acridinium; Zomer G., Van den Berg R.H., Jansen E.H.J.M. (1988) xe2x80x9cOptimal labelling of proteins with acridinium esterxe2x80x9d Anal. Chim. Acia, 205, 267-271.
N-hydroxysuccinimide (NHS) chemistry
A NHS ester is formed by the reaction of a carboxylate with NHS in the presence of a carbodiimide. To prepare a stable ester, the reaction must be done in non-aqueous environments, otherwise it will degrade with non-productive reactions; aqueously prepared esters are unstable and breakdown in a matter of hours under the best of conditions. The NHS ester- or sulf-NHS ester-containing reactant reacts with a nucleophile to form an acylated product with the release of NHS or sulfo-NHS leaving group. The reaction is non-productive with imidazolyl ring nitrogens, sulfhydral or hydroxyl groups, forming aqueously degraded ester or thioester bonds. Reactions with primary and secondary amines create stable amide and imide linkages, respectively.
In proteins these reagents principally react with alpha amines at the N-terminals and the epsilon amines of lysine side chains. It is possible to create NHS esters in situ to react immediately with target molecules in aqueous media (Staros, i., Wright, R. and Swingel, D. (1986), Anal. Biochem., 156, 220-222; Staros, J. (1982), Biochemistry 21, 3950-3955). NHS esters have a half-life in aqueous environments at pH 7 on the order of several hours (4-5 hours at 0xc2x0 C. pH 7.0, Lomant, A. and Fairbanks, G. (1976), J Mol. Biol., 104, 243-261).
Carbodiimides are zero length cross-linkers that mediate the formation of an amide or phosphoramidate linkage between a carboxylate and an amine, or a phosphate and an amine, respectively. (Chu, B., Kramer, F. and Orgel, L. (1986), xe2x80x9cSynthesis of an amplifiable reporter RNA for bioassays,xe2x80x9d Nucleic Acids Research, 14, 5591-5603. Hoare, D. and Koshland, D.E. (1966) J. Am. Chem. Soc., 88, 2057.) They react with carboxylic acids to form highly reactive O-acylisourea compounds that are very short lived but react with nucleophiles to form an amide bond. There are several competing and non-productive reactions, such as with water to regenerate the carboxylate group. This reaction works effectively between pH 4.5 and 7.5. Molecules with a phosphate group such as the 5xe2x80x2 phosphate on oligonucleotides can also react with amine-containing groups by using the carbodiimide reaction.
DCCD (a carbodiimide) and NHS have been used to activate a microplate surface, which was then washed and protein added to the well to facilitate covalent attachment of the protein to the plate. (Dagenais P., Desprez B., Altert J., Escher E. (1994), Anal. Biochem., 222, 149-155)
A method for use of EDAC (a carbodiimide) and sulfo-NHS is summarized in Hermanson, G.T., Bioconjugate Techniques, Academic Press, New York, London, 1966.
A protein/label complex has been made into a NHS derivative such that when it bonded to the protein transporter it would complex there and allow visualization of the position of the transporter. This is basically an activated protein/dye complex that has an active NHS on it. (Fan J., Pope L.E., Vitols K.S., Huennekens F.M. (1991), xe2x80x9cAffinity labeling of folate transport protein with the N-hydroxysuccinimide ester of the g isomer of fluoresein-methotrexate.xe2x80x9d Biochem., 30, 4573-4580]. NHS activated dyes are also sold on a commercial basis (e.g., see the activated Cyanine dyes sold by Amersham Pharmacia Biotech, such as FluoroLink(trademark)Cy5(trademark) or Flurolink(trademark)Cy5.5(trademark)).
A NHS ester of 4-hydroxytestosterone-4-hemiglutarate has been made by treatment with carbodiimide and NHS. This was mixed with horse-radish peroxidase (HRP) or xcex2-galactosidase to make a 4HT-4-HG that was enzyme labeled. The enzyme labeled hydroxytestosterone was used for tracer studies (Hosada H., Karube T., Kobayashi N., Nambara T. (1985), xe2x80x9cEnzyme labeling of steroids by the N-succinimidyl ester method. Preparation of horse-radish peroxidase-labelled antigen for use in enzyme immunoassay.xe2x80x9d Chem. Pharm. Bull., 33, 249-255).
The present invention is directed to simplified methods for coupling labels to particular target moieties. Such coupling is usually accomplished by activating the label, the target moiety, or both with highly reactive activating chemicals. The usual coupling protocols are problematic because the activating chemicals are prone to uncontrolled reactions which produce unwanted side-products, or a too rapid reaction resulting in dissipation of the activating chemicals without the desired coupling. Coupling reactions according to the present invention use temporal spacing of the reactants through phase change (e.g., by rapid freezing) to control the initiation and termination of reaction instead of physical spacing of the traditional method.
For example, the technique of generating NHS esters in situ (with no freeze-drying, just adding in reagents) is mentioned in Bioconjugate Techniques. This invention uses the same chemistry and the same idea of getting a label bound to another molecule, but the difference is the easy-to-use format (i.e., the xe2x80x9cone tube formatxe2x80x9d). In this method, all reactants are prepared separately then combined in such a way that they do not react with each other until the targeted compound is added to activate the cross-linking chemicals. One such format is done by sequentially flash freezing aqueous solutions of the reactants then freeze-drying them together as a unit (e.g., in a microcentrifuge tube). In another format of this invention, freeze-dried or otherwise dried components are prepared separately and then combined in a dry form or in a spatially separate format (e.g., in the top of a microcentrifuge tube that is sectioned into separate parts), such that the water soluble components are not in condition to react with each other until addition of the compound to be labeled (in aqueous form).
This invention provides an improved method of linking labels to specific binding moieties (e.g., moieties that are useful for selecting analytical targets) using N-hydroxysuccinimide (NHS) chemistry. In particular, the invention provides for the separation and stabilization of the active components achieved through sequential or separate freezing in the appropriate buffers (essentially spatial or temporal separation of components prior to utilization).
In another mode, this invention provides a kit comprising NHS, a water soluble carbodiimide (e.g., EDAC) and a label containing an amine or carboxyl moiety, these components being in dry form suitable for rehydration at a pH around 7 in which all components have sufficient activity for activation and cross-linkage.
While phycobiliproteins have been considered complicated to conjugate, this invention provides a method for rapidly and easily making stable phycobiliprotein conjugates to specific target binding proteins. SMCC and SPDP pre-activated phycobiliproteins have been available in several formulations. However, this invention provides a simpler method for making conjugates in that it requires fewer steps and is more rapid. In fact, this invention allows a researcher to easily label whatever specific binding molecule they would like with a carboxyl-containing label (e.g., phycobiliprotein, enzyme, PBXL(trademark), etc.) This invention provides for kits to make a conjugate, so that the person using the kit does not have to be an expert at protein conjugation to obtain useable conjugates at the end of the process.